Delineation of the functional properties and the mechanism of action of AA29504, an allosteric agonist and positive allosteric modulator of GABA receptors.

The retigabine analog 2-amino-4-[(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester (AA29504) is a positive allosteric modulator (PAM) of γ-aminobutyric acid receptors (GABARs), and the modulator has been used in ex vivo/in vivo studies to probe the physiological roles of native δ-containing GABARs. In this study, the functional properties and mode of action of AA29504 were investigated at human GABARs expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology. AA29504 was found to be an allosteric GABAR agonist displaying low intrinsic activities at 3-30 μM. AA29504 was essentially equipotent as a PAM at the 13 GABAR subtypes tested (EC: 0.45-5.2 μM), however GABA EC-evoked currents through αβδ subtypes were modulated to substantially higher levels than those through αβγ subtypes (relative to GABA I). While the δ/γ-difference clearly was key for this differential GABA efficacy modulation, studies of the AA29504-mediated modulation of different α-containing αβ, αβγ and αβδ GABARs revealed the α-subunit identity to be another important determinant. Based on its functional properties at numerous mutant GABARs and on in silico analysis of its low-energy conformations, AA29504 is proposed to act through an allosteric site in the transmembrane β/α interface in the GABAR also targeted by etomidate and several other modulators. In contrast to these modulators, however, AA29504 did not display substantial β/β-over-β GABAR preference, which challenges the notion of ligands targeting this site always possessing this subtype-selectivity profile. Hence, the detailed pharmacological profiling of AA29504 both highlights the complexity of allosteric GABAR modulation and provides valuable information about this modulator as a pharmacological tool.